Heme-Containing Oxygenases

Two decades have passed since the discovery in liver microsomes of a haemprotein that forms a reduced-CO complex with the absorptive maximum of the Soret at 450 nm (Klingenberg, 1958; Garfinkel, 1958) and the identification of this protein as a new cytochrome: pigment cytochrome, P-450 (Omura and Sato, 1962, 1964a). In the intervening years, the study of cytochrome P-450 dependent monoxygenases has expanded exponentially. From the first crude attempts to solubilise a P-450 (Omura and Sato, 1963, 1964b) to the determination of the primary, secondary, and tertiary structure of cytochrome P-450cam by amino acid sequencing (Haniu et al., 1982a,b) and x-ray crystallography (Poulos et al., 1984) our understanding of this unique family of proteins has been advancing on all fronts. Since, perhaps, the greatest understanding of the structure and mechanism of P-450s has come from concentrated study of P-450cam of the Pseudomonas putida camphor-5-exo-hydroxylase, this review will concentrate on findings with P-450cam; attention will be drawn to parallel and contrasting examples from other P-450s as appropriate.

Structure and Chemistry of Cytochrome P450

Cholesterol and Bile Acid Metabolism Are Impaired in Mice Lacking the Nuclear Oxysterol Receptor LXRα

Oxygenated derivatives of cholesterol (oxysterols) present a remarkably diverse profile of biological activities, including effects on sphingolipid metabolism, platelet aggregation, apoptosis, and ...

Oxysterols: modulators of cholesterol metabolism and other processes.

LXRα and -β are nuclear receptors that regulate the metabolism of several important lipids, including cholesterol and bile acids. Previously, we have proposed that LXRs regulate these pathways through their interaction with specific, naturally occurring oxysterols, including 22(R)-hydroxycholesterol, 24(S)-hydroxycholesterol, and 24(S),25-epoxycholesterol. Using a ligand binding assay that incorporates scintillation proximity technology to circumvent many of the problems associated with assaying extremely hydrophobic ligands, we now demonstrate that these oxysterols bind directly to LXRs at concentrations that occur in vivo. To characterize further the structural determinants required for potent LXR ligands, we synthesized and tested a series of related compounds for binding to LXRs and activation of transcription. These studies revealed that position-specific monooxidation of the sterol side chain is requisite for LXR high-affinity binding and activation. Enhanced binding and activation can also be achieved through the use of 24-oxo ligands that act as hydrogen bond acceptors in the side chain. In addition, introduction of an oxygen on the sterol B-ring results in a ligand with LXRα-subtype selectivity. These results support the hypothesis that naturally occurring oxysterols are physiological ligands for LXRs and show that a rational, structure-based approach can be used to design potent LXR ligands for pharmacologic use.

https://www.pnas.org/content/pnas/96/1/266.full.pdf

Structural requirements of ligands for the oxysterol liver X receptors LXRalpha and LXRbeta.

A double isotope derivative method for the estimation of progesterone in 10 ml peripheral plasma, using 35S-thiosemicarbazide (100 mc/mM) as reagent and 1,2-3H-progesterone (0.8 mμg, 87 μc/μg) a s indicator, is described. Purification is achieved by chromatography of the first derivative formed, progesterone bisthiosemicarbazone, by a silica gel thin layer and a paper system, hydrolysis to progesterone 3-thiosemicarbazone and chromatography of this derivative by a thin layer system, and finally acetylation to the 3-thiosemicarbazone 2′,4′-diacetate, and subsequent chromatography of this derivative on one thin layer and one paper system. The values given by 10 ml water samples and the same volume of plasma from ovariectomized, adrenalectomized subjects were 0.0020 ±0.0009 (sd) ±0.0002 (se) μg (23 samples) and 0.0029 ±0.0013 (sd) ±0.006 (se) μg (5 samples), respectively. These mean values were therefore the nonspecific blank of the method. No known naturally occurring steroids were found to interfere with t...

Estimation of Progesterone in Human Peripheral Blood Using 35S-Thiosemicarbazide

20α, 22ξ-Dihydroxycholesterol, an Intermediate in the Biosynthesis of Pregnenolone (3β-Hydroxypregn-5-en-20-one) from Cholesterol

The formation of estrogens and phenolic material from a large number of do Cis, C19 and C2i steroids and related structures has been investigated, employing a human placental microsomal preparation supplemented with TPNH and oxygen. The data from the incubation of 2 C 14-labeled androgens are included. There is a discussion of possible biosynthetic pathways for the estrogens.

Marcel Gut

Papers

Estimation of Progesterone in Human Peripheral Blood Using 35S-Thiosemicarbazide

20α, 22ξ-Dihydroxycholesterol, an Intermediate in the Biosynthesis of Pregnenolone (3β-Hydroxypregn-5-en-20-one) from Cholesterol

Biosynthesis of estrogens.

The Transformation of 20α-Hydroxycholesterol to Isocaproic Acid and C21 Steroids

Steric considerations in the enzymatic course of the hydroxylation of steroids